Cellular resistance is the major obstacle to the successful treatment of disseminated malignancies. A fundamental resistance mechanism involves reducing cellular drug concentrations by pumping cytotoxic agents across the plasma membrane. P-glycoprotein, an ABC family transporter that confers resistance to several natural product drugs, was the first example of this mechanism. More recently MRP, an ABC transporter distantly related to Pgp, has been shown to confer a multidrug resistance phenotype that is similar but not identical to Pgp. In addition, increasing evidence indicates that a close relative of MRP, the canalicular multispecific organic anion transporter (cMOAT), confers resistance to both natural product drugs and cisplatin in cells in which it is induced. cMOAT, whose normal expression is restricted to liver canaliculi, has also been demonstrated to be involved in the hepatobiliary excretion of anticancer drugs such as methotrexate, by pumping these agents directly into the bile. Our laboratory has recently isolated four novel MRP/cMOAT-related transporters, designated MOAT-B, C, D and E. These four transporters, along with MRP and cMOAT, form a tight evolutionary cluster that defines the MRP/cMOAT subfamily. Analysis of the predicted structures of these four transporters reveals that D is the closest known relative of MRP (significantly more related to MRP than is cMOAT), and that E and cMOAT are about equally related to MRP. Expression of MOAT-D is primarily restricted to liver, kidney, colon and pancreas, and E is expressed only in the kidney and liver. These observations are highly suggestive that D and E are involved in both cellular resistance as well as the hepatobiliary and renal excretion of anticancer agents. While MOAT-B and MOAT-C are not as closely related to MRP as are D and E, they are part of the MRP/cMOAT evolutionary cluster, and may therefore also be involved in cellular resistance. The purpose of this proposal is to elucidate the roles of these novel MRP/cMOAT-related transporters in cellular resistance and hepatobiliary and renal excretion of anticancer agents. Elucidating their function will provide important information concerning the drug resistant phenotype and the pharmacokinetics of anticancer agents, and will impact the development of modulation strategies designed to circumvent membrane-based resistance mechanisms.